_Archives
Vi rology
Arch Virol (1992) 126:45-56
© Springer-Verlag 1992 Printed in Austria
Analysis of the genetic diversity of genes 5 and 6 among group C rotaviruses using c D N A probes B. M. Jiang 1, H. Tsunemitsu1, Y. Qian 2, K. Y. Green2, M. Oseto 3, Y. Yamashita ~, and Linda J. Saif 1 1Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 2Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. 3Ehime Prefectural Institute of Public Health, Matsuyama, Ehime, Japan Accepted February 17, 1992
Summary. Two partial eDNA clones of genes 5 (encoding the major inner capsid protein VP 6) and 6 (encoding a nonstructural protein) of the porcine group (Gp) C rotavirus (Cowden strain) were radiolabeled with 32p and used individually as probes in Northern and dot blot hybridization assays. The specificity of each probe was tested against genomic dsRNA from: (1) porcine Gp A, B, and C rotaviruses; (2) Gp C rotaviruses from different species; and (3) porcine Gp C rotavirus field strains with varying electropherotype patterns. Neither probe hybridized with ds RNA from the porcine Gp A and B strains under the stringency conditions employed in the study. However, the gene 5 probe hybridized with the corresponding gene from the homologous porcine and the heterologous human and bovine Gp C rotaviruses tested. The gene 6 probe hybridized with the corresponding gene from the homologous Cowden strain, but hybridized weakly with gene 6 from the human and bovine Gp C rotaviruses. Both probes recognized all six different porcine Gp C field strains, although with varying intensities. Our results demonstrate that the gene 5 and 6 probes used in this study are specific for Gp C rotaviruses. However, evidence for greater genetic variation in the gene 6 among porcine, bovine and human Gp C strains suggested that the gene 5 probe may prove more broadly reactive among Gp C strains from different species, cDNA probes used in our study should prove useful for the detection of Gp C rotaviruses in feces and facilitate epidemiologic studies. Introduction Rotaviruses, members of the family Reoviridae, are double-shelled viral particles that are associated with diarrheal disease in humans and animals [-11, 30]. To
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date, seven serotogically distinct groups (A to G) of rotaviruses have been identified and characterized [4, 30]. Among them, Gp C rotaviruses were originally isolated from diarrheic pigs in the U.S. [31] and infants in Australia, Europe and Mexico [-7, 10, 21, 28]. Since then, further outbreaks of diarrhea associated with human Gp C rotaviruses have been reported in Asia and Europe [-5, 19, 24, 39]. Because of the inability to routinely propagate most Gp C rotaviruses in cell culture, it is important that other reagents be developed for epidemiologicstudies of Gp C rotavirus so that the extent of its role in diarrheal disease of both humans and animals can be established. Gp C rotaviruses are genetically and antigenically distinct from the Gp A rotaviruses [1-3, 9, 13, 14]. Within Gp C rotaviruses, porcine and human strains share common antigenic and genetic characteristics, as demonstrated by serologic [24], hybridization [14, 25], and sequence studies [6, 26]. The prototype porcine Gp C Cowden strain has been adapted to growth in cell culture [32] and serves as an important model for studying other noncultivatable Gp C rotaviruses. In Gp A rotaviruses, genes 5 and 6 encode a nonstructural protein (NS 53) and the inner capsid protein (VP 6) which carries the group or subgroup antigens, respectively [11, 18]. In contrast, recent studies have demonstrated that genes 5 and 6 of Gp C rotaviruses code for the structural protein VP 6 and the NS 34 equivalent of Gp A rotavirus, respectively [6, 27]. Thus, gene 5 and 6 cDNA clones were selected from a Cowden Gp C cDNA library and were developed for use as radiolabeled cDNA probes. The specificity of these two probes was examined against representative rotaviruses from different groups (A, B, or C), Gp C rotaviruses from different species, and distinct field strains of porcine Gp C rotaviruses in order to analyze the genetic diversity of genes 5 and 6 among Gp C rotaviruses and to develop cDNA probes for use as diagnostic and epidemiologic reagents.
Materials and methods Viruses
Human Gp C rotavirus strain 88-196 was purified from stool material collected from a 15-year-oldfemalewith diarrhea in Japan. BovineGp C rotavirus strain Shintoku, isolated from a diarrheic cow in Japan and adapted to growth in an MA 104 monkey kidney cell line [38], was purified from tissue culture material. Porcine Gp C rotavirus strain Cowden was purified from the intestinal contents of experimentally infected gnotobiotic pigs as previously described [-1]. Six porcine Gp C field strains were derived from the stools of diarrheic pigs in Ohio (NB, WH, Ah, HF, and KH) and Mississippi (Wi). An intestinal filtrate of each field strain was passaged in gnotobiotic pigs and the intestinal contents collectedasepticallyat the onset of diarrhea [36]. Two additional porcine rotavirus strains were included in the study: Gottfried (Gp A), propagated in MA 104 cells, and Ohio (Gp B), passaged in gnotobiotic pigs [37]. An uninfectedMA 104 cell extract and a fecal sample from an uninfected gnotobiotic pig were used as controls.
Genetic diversity of genes 5 and 6 among group C rotaviruses
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Preparation of viral dsRNA Samples containing virus particles were homogenized in Tris-CaC12 buffer (0.05 M Tris, 0.1 M NaC1, 1.0 mM CaC12, pH 7.5). The mixture was clarified by centrifugation at 8.700 x g and the resulting supernatant was centrifuged through a 30% (w/v) sucrose cushion at 107.170 x g for 2 h at 4 °C. The virus pellet was suspended in Tris-sodium chloride-EDTA buffer (0.01M Tris, 0.1M NaC1, 5raM EDTA, pH8.0) and SDS was added to a final concentration of 1%. The mixture was extracted with phenol-chloroform and the dsRNA precipitated with 2 volumes of ethanol overnight at - 20 °C. The concentration of nucleic acid was calculated from the absorbance at 260 nm. The two negative control samples were processed in the same manner.
Preparation of eDNA probes The generation and characterization of the cDNA clones of the Cowden genes 5 and 6 used in this study have been described [27]. Clone PC 4-19 consisted of nucleotides 356 to 1,352 of the Cowden gene 5 and clone CA2-19 consisted of nucleotides 115 to 1,145 of the Cowden gene 6 inserted into plasmid pTZ 18 R (Pharmacia LKB Biot~hnology Inc., Piscataway, N J). The recombinant DNA was nick-translated (Bethesda Research Laboratories. Inc., Gaithersburg, MD) using [32 p] dCTP (ICN Biochemicals Inc., Irvine, CA). The unincorporated nucleotides were removed by centrifugation through a Sephadex G-50 column [33].
RNA electrophoresis Viral dsRNA (1 to 5 gg per lane) was resolved in a 10% polyacrylamide slab gel (0.75 mm thick) using Laemmli's discontinuous buffer system [15]. Electrophoresis was conducted at 10 to 12mA for 14h. The RNA bands were visualized by ethidium bromide staining (0.5 gg/ml).
Northern and dot blot hybridization Northern blot hybridization was conducted using a method previously described [35]. Briefly, viral RNA in potyacrylamide gels was denatured in 0.1 M NaOH for 20 rain. The soaked gels were washed twice in 4 x TAE (1 x is 0.04 M Tris-acetate and 0.001 M EDTA) and once in 1 x TAE. The denatured RNA was then electro-transferred onto Nytran membranes (Schleicher & Schuell, Keene, NH) and immobilized by exposure to UV light. Hybridization conditions were similar to those previously described [29]. Prehybridization was for 4h at 42 °C in a solution containing 50% formamide, 5 x SSC, 50mM phosphate buffer (pH6.5), 0.2% SDS, 2 x Denhardt's solution, and 100gg/ml of yeast tRNA. Unless otherwise stated, all hybridizations were performed at 42 °C for 16 to 24 h in a similar solution with the addition of 4.5% dextran sulfate and the radiolabeled DNA probe (10 6 CPM/ml; specific activity, 650 Ci/mmote). Subsequently, the membranes were washed four times at room temperature in 2 x SSC, 0.1% SDS and then twice at 42 °C in 0.4 x SSC, 0.1% SDS. Finally, the membranes were exposed to Kodak diagnostic films (X-Omat RP-1; Eastman Kodak Company, Rochester, NY) at - 70°C for 1 to 6 days. For dot hypridization, heat-denatured RNA (0.5 to 1.0 gg) was dotted onto the Nytran membranes. The hybridization assay was conducted as described above.
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Jiang et al.: Genetic diversity of genes 5 and 6 among group C rotaviruses Results
Northern blot hybridization of Cowden strain gene 5 and 6 cDNA probes with Gp A, B and C rotaviruses The group specificity of the cDNA probes was examined by Northern blot hybridization. The distinct RNA electropherotypes of Gp A, B and C rotaviruses can be seen in Fig. 1 a and c. The Cowden Gp C gene 5 and 6 probes each recognized the corresponding gene from the Cowden strain, the excised homologous cDNA insert and the pTZ 18 R plasmid (Fig. 1 b and d, lanes 1 and 2). Neither probe hybridized with dsRNA from the Gp A or B rotaviruses under both moderate stringency conditions (50% formamide, 5 x SSC and 42 °C) (Fig. 1 b and d, lanes 3 and 4) and low stringency conditions (25% formamide, 5 x SSC, and 42 °C) (data not shown).
Northern blot hybridization analysis of Cowden gene 5 and 6 probes with Gp C rotaviruses from different species Northern blot hybridization was utilized to examine the species specificity of the gene 5 and 6 probes with Gp C rotaviruses from pigs, cows and humans. The similarities of RNA electropherotypes from Gp C rotaviruses of human and animal origin are seen in Fig. 2 a and c. In addition to hybridization with the excised homologous cDNA insert and the plasmid (Fig. 2b, lane 1), the gene 5 probe reacted with genomic segment 5 of Gp C rotaviruses from all three species, although relatively weaker signals were observed against the bovine and human Gp C rotaviruses (Fig. 2 b, lanes 2-4). In contrast, the gene 6 probe reacted strongly with the homologous Cowden gene 6, but signals were hardly detected with the gene 6 of bovine and human Gp C rotaviruses (Fig. 2 d, lanes 2-4) (weak signals could be seen after prolonged autoradiography; data not shown). However, at the low stringency conditions, hybridization signals were apparent with gene 6 of bovine and human Gp C rotaviruses (data not shown).
Northern blot hybridization of Cowden strain gene 5 and 6 probes against field strains of porcine Gp C rotavirus The strain specificity of the gene 5 and 6 probes was examined by Northern blot hybridization. In comparison with the prototyope Cowden strain, variation in the RNA electrophoretic migration patterns of the six field strains is seen although the overall RNA etectropherotype pattern is characteristic for Gp C rotavirus (Fig. 3 a and c). Each probe hybridized with the corresponding gene 5 or 6 of all field strains tested. However, the intensity of the signals on the autoradiogram varied. For example, the gene 5 probe showed strong signals with the gene 5 of the Cowden, NB, and Ah strains, moderate signals with the WH, HF, and Wi strains, and a weak signal with the KH strain (Fig. 3 b). With the gene 6 probe, strong signals were seen with the gene 6 of the Cowden, WH,
Fig. 1. Northern blot hybridization analysis of Cowden strain gene 5 (clone PC4-19) and 6 (clone CA 2-19) cDNA probes against representative porcine Gp A and B rotaviruses. Viral dsRNA was resolved in 10% polyacrylamide gel using Laemmli's discontinuous buffers and transferred onto Nytran membranes as described in the text. The hybridization was conducted at moderate stringency conditions (50% formamide, 5 x SSC and 42 °C). a and c Ethidium bromide-stained plasmid DNA and cDNA inserts (1 eDNA inserts were excised from recombinant DNA by digesting with EcoRI and BamHI and separated from the plasmid DNA), and RNA electropherotypes of Gp C (2), A (3) and B (4) rotaviruses. b and d The corresponding autoradiogram using the 32P-labeled gene 5 and 6 probes as indicated
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Fig. 2. Northern blot hybridization analysis of Cowden strain gene 5 and 6 probes against Gp C rotaviruses from different species. Samples were analyzed as described in the legend to Fig. 1. a and e Ethidium bromide-stained plasmid DNA and cDNA inserts (1), and RNA electropherotypes of porcine (2), bovine (3) and human (4) Gp C rotaviruses, b and d The corresponding autoradiogram using the 32P-labeled gene 5 and 6 probes as indicated
and Wi strains, moderate signals with the NB and K H strains, and weak signals with the Ah and H F strains (Fig. 3 d). Some weak signals could be attributed to lower d s R N A concentrations (e.g., the gene 5 probe against the K H strain), whereas other weak signals could not be attributed to lower R N A concentrations (e.g., the gene 5 probe against the W H strain).
Genetic diversity of genes 5 and 6 among group C rotaviruses
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Fig. 3. Northern blot hybridization of porcine Gp C Cowden strain gene 5 and 6 probes against field strains of porcine Gp C rotavirus. Samples were analyzed as in Fig. 1 a and e Ethidium bromide-stained plasmid DNA and cDNA inserts (DNA), and RNA migration patterns of the Cowden (Co) strain and 6 field strains (NB, WH, Ah, HF, KH, and Wi). b and d The corresponding autoradiogram using the 32P-labeled gene 5 and 6 probes as indicated
Analysis by dot hybridization The results of d o t hybridization assays were similar to N o r t h e r n blot hybridization using the same probes and stringency conditions. The gene 5 and 6 probes each hybridized with d s R N A from the h o m o l o g o u s G p C C o w d e n strain, but neither hybridized with d s R N A from the G p A Gottfried strain or the G p
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B Ohio strain (Fig. 4, 1 and 1 a). Against Gp C rotaviruses from three different species (porcine, bovine, and human), both probes recognized the homologous and heterologous viruses (Fig. 4, 2 and 2 a). However, similar to the Northern blot hybridization, much weaker signals were detected against the heterologous Shintoku and 88-196 strains with the gene 6 probe (Fig. 4, 2 a). Both probes hybridized with d s R N A from all six Gp C field strains in the dot blots. However, a variation in the intensities of the signals on the autoradiogram was observed. For example, weak signals were detected with the gene 5 probe against the H F and KH strains and with the gene 6 probe against the Ah and H F strains (Fig. 4, 3 and 3 a). Both probes hybridized strongly with the corresponding
Fig. 4. Dot hybridization analysis of Cowden gene 5 (1-3) and 6 (1 a-3 a) probes against Gp C, A, and B rotaviruses (1, i a); porcine, bovine, and human Gp C rotaviruses (2, 2 a); and homologous (Cowden) and heterologous strains of porcine Gp C rotaviruses designated NB, WH, Ah, HF, KH, and Wi (3, 3 a). The positive (recombinant DNA PC 4-19 or CA 219) and negative (MA 104 cells and porcine fecal specimen) controls are also included. Samples were dotted onto Nytran membranes and hybridized with 32P-labeled gene 5 and 6 probes as described in the text
Genetic diversity of genes 5 and 6 among group C rotaviruses
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undigested recombinant DNA, but no signal was detected against an uninfected MA 104 cell extract or a negative porcine fecal specimen (Fig. 4, 1 and 1 a). The sensitivity of the two probes was investigated by dot hybridization against serial dilutions of Cowden dsRNA. Each probe was capable of detecting as low as 7.8 ng of RNA (data not shown). Discussion
Several criteria have been established to define rotavirus groups, including lack of serologic cross-reactivities among group antigens using heterologous antisera, RNA etectropherotyping and terminal fingerprinting of individual genomic segments [22, 23, 30]. Hybridization assays have been successfully used in the diagnosis of Gp A rotavirus infections, the analysis of the genetic relatedness among Gp A rotaviruses and the differentiation of Gp A from non Gp A rotaviruses [8, 9, 12, 16, 20, 29]. Further, cDNA probes derived from the gene encoding VP 6 (gene 6) of the Gp A Wa (subgroup II) and SA 11 (subgroup I) strains were used for differentiating Gp A rotaviruses into subgroups [ 17]. This genetic approach for subgrouping Gp A rotaviruses correlated with serologic analysis using subgroup-specific monoclonal antibodies. The cDNA gene 5 probe developed here proved to be Gp C rotavirus-specific, while reacting broadly among Gp C rotaviruses from different species. The variable reactivities of our gene 5 probe against dsRNA of similar quantity among field strains of porcine Gp C rotaviruses might reflect subgroup differences. However, no antigenic evidence for subgroup differences among Gp C rotaviruses has yet been reported. The gene 6 probe successfully recognized several distinct porcine Gp C rotavirus field strains, while demonstrating Gp C rotavirus-specificity. An interesting observation in this study was the apparently greater genetic diversity among the gene 6 of Gp C rotavirus strains from different species as detected by the intensity of the hybridization signal on the autoradiogram. Similar studies of genetic diversity among Gp A rotaviruses from different species using cDNA probes to genes encoding a nonstructural protein have not been reported, although evidence for genetic diversity in a gene encoding a nonstructural protein (gene 5) was found among human Gp A rotaviruses in RNA-RNA hybridization studies [34]. The biological significance of the genetic variation among genes 5 and 6 of Gp C strains requires further analysis of additional strains. Recently, the genetic relatedness of human (88-220 strain) and porcine (Cowden strain) Gp C rotaviruses was examined by Northern blot hybridization analysis using ssRNA probes, and strong homology was observed in most of the 11 gene segments [25]. Our findings are consistent with that report. Our previous study reported the genetic relatedness of the gene encoding the outer capsid protein (VP 7) between porcine (Cowden strain) and bovine (Shintoku strain) Gp C rotaviruses using the VP 7 cDNA probe [14]. This study further demonstrated the genetic relatedness of genes 5 and 6 between porcine and
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bovine Gp C rotaviruses. The identification of a group C rotavirus in cattle indicates that, analogous to Gp A rotaviruses, Gp C rotaviruses m a y exhibit a broad host range. The unavailability of reagents for the identification of Gp C rotaviruses a n d the inability to serially p r o p a g a t e m o s t strains in cell culture have h a m p e r e d epidemiologicat surveys. In this study, we report the development o f hybridization assays for the identification o f G p C rotaviruses using the C o w d e n gene 5 and 6 c D N A probes. These assays should provide sensitive and specific m e t h o d s for the diagnosis of G p C rotavirus infections in h u m a n s a n d animals and should facilitate epidemiological studies. However, for large scale epidemiologic studies, nonradiolabeled probes m a y be preferable for routine use. Acknowledgements
We thank A. V. Parwani and B. I. Rosen for assistance in the hybridization assays. This study was supported in part by special research grant #89-34116-4625 from the U.S. Department of Agriculture, Cooperative State Research Service. Salaries and research support were provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Journal article No. 66-91. References
1. Bohl EH, Saif LJ, Theil KW, Agnes AG, Cross RF (1982) Porcine pararotavirus: detection, differentiation from rotavirus, and pathogenesis in gnotobiotic pigs. J Clin Microbiol 15:312-319 2. Bremont M, Cohen J, McCrae MA (1988) Analysis of the structural polypeptides of a porcine group C rotavirus. J Virol 62:2183-2185 3. Bremont M, Chabanne-Vautherot D, Vannier P, McCrae MA, Cohen J (1990) Sequence analysis of the gene (6) encoding the major capsid protein (VP 6) of group C rotavirus: higher than expected homology to the corresponding protein from group A virus. Virology 178:579-583 4. Bridget JC (1987) Novel rotaviruses in animals and man. CIBA Found Symp 128: 523 5. Caul EO, Ashley CR, Darville JM, Bridger JC (1990) Group C rotavirus associated with fatal enteritis in a family outbreak. J Med Virot 30:201-205 6. Cooke SJ, Lambden PR, Caul EO, Clarke IN (1991) Molecular cloning, sequence analysis and coding assignment of the major inner capsid protein gene of human group C rotavirus. Virology 184:781-784 7. Dimitrov DH, Estes MK, Rangelova SM, Shindarov LM, Melnick JL, Graham DY (1983) Detection of antigenically distinct rotaviruses from infants. Infect Imun 41: 523526 8. Dimitrov DH, Graham DY, Estes MK (1985) Detection of rotaviruses by nucleic acid hybridization with cloned DNA of simian rotavirus SA 11 genes. J Infect Dis 152: 293300 9. Eiden J, Vonderfecht S, Theil K, Torres-Medina A, Yolken RH (1986) Genetic and antigenic relatedness of human and animal strains of antigenically distinct rotaviruses. J Infect Dis 154:972-982 t0. Espejo RT, Puerto F, Soler C, Gonzalez N (1984) Characterization of a human pararotavirus. Infect Immun 44:112-116 11. Estes MK, Cohen J (1989) Rotavirus gene structure and function. Microbiol Rev 53: 410-449
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12. Flores J, Perez-Schael I, Boeggeman E, White L, Perez M, Purcell R, Hoshino Y, Midthun K, Chanock RM, Kapikian AZ (1985) Genetic relatedness among human rotaviruses. J Med Virol 17:135-143 13. Jiang BM, Saif LJ, Kang SY, Kim JH (1990) Biochemical characterization of the structural and nonstructural polypeptides of a porcine group C rotavirus. J Virol 64: 3171-3178 14. Jiang BM, Qian Y, Tsunemitsu H, Green KY, Saif LJ (1991) Analysis of the gene encoding the outer capsid glycoprotein (VP 7) of group C rotaviruses by' Northern and dot blot hybridization. Virology 184:433-436 15. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T 4. Nature 227:680-685 16. Lin M, Imai M, Bellamy AR, Ikegami N, Furuichi Y, Summers D, Nuss DL, Deibel R (1985) Diagnosis of rotavirus infection with cloned cDNA copies of viral genome segments. J Virol 55:509-512 17. Lin M, Imai M, Ikegami N, Bellamy AR, Summers D, Nuss DL, Deibel R, Furuichi Y (1987) cDNA probes of individual genes of human rotavirus distinguish viral subgroups and serotypes. J Virol Methods 15:285-289 18. Mason BB, Graham DY, Estes MK (1983) Biochemical mapping of the simian rotavirus SA 11 genome. J Virol 46:413423 19. Matsumoto K, Hatano M, Kobayashi K, Hasegawa A, Yamazaki S, Nakata S, Chiba S, Kimura Y (1989) An outbreak of gastroenteritis associated with acute rotaviral infection in schoolchildren. J Infect Dis 160:611-615 20. Nakagomi O, Oyamada H, Nakagomi T (1989) Use of alkaline northern blot hybridization for the identification of genetic relatedness of the fourth gene of rotaviruses. Mol Cell Probes 3:263-271 21. Nicolas JC, Cohen J, Fortier B, Lourenco MH, Bricout F (1983) Isolation of a human pararotavirus. Virology 124:181-184 22. Pedley S, Bridger JC, Brown JF, McCrae MA (1983) Molecular characterization of rotaviruses with distinct group antigens. J Gen Virol 64:2093-2101 23. Pedley S, Bridget JC, Chasey D, McCrae MA (1986) Definition of two new groups of atypical rotaviruses. J Gen Virol 67:131-137 24. Pefiaranda ME, Cubitt WD, Sinarachatanant P, Taypor DN, Likanonsakul S, Saif L, Glass RI (1989) Group C rotavirus infections in patients with diarrhea in Thailand, Nepal, and England. J Infect Dis 160:392-397 25. Qian Y, Saif LJ, Kapikian AZ, Kang SY, Jiang B, Ishimaru Y, Yamashita Y, Oseto M, Green KY (1991) Comparison of human and porcine group C rotaviruses by Northern blot hybridization analysis. Arch Virol 118:269-277 26. Qian Y, Jiang B, Saif L J, Kang SY, Ishimaru Y, Yamashita Y, Oseto M, Green KY (1991) Sequence conservation of gene 8 between human and porcine group C rotaviruses and its relationship to the VP7 gene of group A rotaviruses. Virology 182: 562-569 27. Qian Y, Jiang B, Saif LJ, Kang SY, Green KY (1991) Molecular analysis of the gene 6 from a porcine group C rotavirus that encodes the NS 34 equivalent of group A rotaviruses. Virology 184:752-757 28. Rodger SM, Bishop RF, Holmes IH (1982) Detection of a rotavirus-like agent associated with diarrhea in an infant. J Clin Microbiol 16:724-726 29. Rosen BI, Saif LJ, Jackwood DL, Gorziglia M (1990) Serotypic differentiation of group A rotaviruses with porcine rotavirus gene 9 probes. J Clin Microbiol 28:2526 2533 30. Saif LJ (1990) Nongroup A rotaviruses. In: LJ Saif, Theil KW (ed) Viral diarrheas of man and animals. CRC Press, Boca Raton, pp 73-95 31. Saif LJ, Bohl EH, Theil KW, Cross RF, House JA (1980) Rotavirus-like, calcivirus-
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Jiang et al.: Genetic diversity of genes 5 and 6 among group C rotaviruses like, and 23-nm virus like particles associated with diarrhea in young pigs. J Clin Microbiol 12:t05-111 Saif LJ, Terrett LA, Miller KL, Cross RF (1988) Serial propagation of procine group C rotavirus (pararotavirus) in a continuous cell line and characterization of the passaged virus. J Clin Microbiol 26:1277-1282 Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning, a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring Harbor, pp E. 34-E. 38 Stree JC, Croxson MC, Chadderton WF, Bellamy AR (1982) Sequence diversity of human rotavirus strains investigated by Northern blot hybridization analysis. J Virol 43:369-378 Tanaka TN, Conner ME, Graham DY, Estes MK (1988) Molecular characterization of three rabbit rotavirus strains. Arch Virol 98:253-265 Terrett LA, Saif LJ, Theii KW, Kohler EM (1987) Physicochemical characterization of porcine pararotaviruses and detection of viral antibodies using cell culture immuno fluorescence. J Clin Microbiol 25:268-272 Theit KW, Saif L J, Moorhead PD, Whitmoyer RE (1985) Porcine rotavirus-like virus (group B rotavirus): characterization and pathogenicity for gnotobiotic pigs. J Clin Microbiol 21:340-345 Tsunemitsu H, Saif LJ, Jiang B, Shimizu M, Masanobu H, Yamaguchi T, Ishiyama T, Hirai T (1991) Isolation, characterization and serial propagation of a bovine group C rotavirus in a monkey kidney (MA 104) cell line. J Clin Microbiol 29:2609-2613 Ushijima H, Honma H, Mukoyama A, Shinozaki T, Fujita Y, Kobayashi M, Oseto M, Morikawa S, Kitamura T (1989) Detection of group C rotavirus in Tokyo. J Med Virol 27:299-303
Authors' address: Linda J. Saif, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691-4096, U.S.A. Received November 24, 1991
Vi rology
Arch Virol (1992) 126:45-56
© Springer-Verlag 1992 Printed in Austria
Analysis of the genetic diversity of genes 5 and 6 among group C rotaviruses using c D N A probes B. M. Jiang 1, H. Tsunemitsu1, Y. Qian 2, K. Y. Green2, M. Oseto 3, Y. Yamashita ~, and Linda J. Saif 1 1Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 2Laboratory of Infectious Diseases, National Institutes of Health, Bethesda, Maryland, U.S.A. 3Ehime Prefectural Institute of Public Health, Matsuyama, Ehime, Japan Accepted February 17, 1992
Summary. Two partial eDNA clones of genes 5 (encoding the major inner capsid protein VP 6) and 6 (encoding a nonstructural protein) of the porcine group (Gp) C rotavirus (Cowden strain) were radiolabeled with 32p and used individually as probes in Northern and dot blot hybridization assays. The specificity of each probe was tested against genomic dsRNA from: (1) porcine Gp A, B, and C rotaviruses; (2) Gp C rotaviruses from different species; and (3) porcine Gp C rotavirus field strains with varying electropherotype patterns. Neither probe hybridized with ds RNA from the porcine Gp A and B strains under the stringency conditions employed in the study. However, the gene 5 probe hybridized with the corresponding gene from the homologous porcine and the heterologous human and bovine Gp C rotaviruses tested. The gene 6 probe hybridized with the corresponding gene from the homologous Cowden strain, but hybridized weakly with gene 6 from the human and bovine Gp C rotaviruses. Both probes recognized all six different porcine Gp C field strains, although with varying intensities. Our results demonstrate that the gene 5 and 6 probes used in this study are specific for Gp C rotaviruses. However, evidence for greater genetic variation in the gene 6 among porcine, bovine and human Gp C strains suggested that the gene 5 probe may prove more broadly reactive among Gp C strains from different species, cDNA probes used in our study should prove useful for the detection of Gp C rotaviruses in feces and facilitate epidemiologic studies. Introduction Rotaviruses, members of the family Reoviridae, are double-shelled viral particles that are associated with diarrheal disease in humans and animals [-11, 30]. To
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date, seven serotogically distinct groups (A to G) of rotaviruses have been identified and characterized [4, 30]. Among them, Gp C rotaviruses were originally isolated from diarrheic pigs in the U.S. [31] and infants in Australia, Europe and Mexico [-7, 10, 21, 28]. Since then, further outbreaks of diarrhea associated with human Gp C rotaviruses have been reported in Asia and Europe [-5, 19, 24, 39]. Because of the inability to routinely propagate most Gp C rotaviruses in cell culture, it is important that other reagents be developed for epidemiologicstudies of Gp C rotavirus so that the extent of its role in diarrheal disease of both humans and animals can be established. Gp C rotaviruses are genetically and antigenically distinct from the Gp A rotaviruses [1-3, 9, 13, 14]. Within Gp C rotaviruses, porcine and human strains share common antigenic and genetic characteristics, as demonstrated by serologic [24], hybridization [14, 25], and sequence studies [6, 26]. The prototype porcine Gp C Cowden strain has been adapted to growth in cell culture [32] and serves as an important model for studying other noncultivatable Gp C rotaviruses. In Gp A rotaviruses, genes 5 and 6 encode a nonstructural protein (NS 53) and the inner capsid protein (VP 6) which carries the group or subgroup antigens, respectively [11, 18]. In contrast, recent studies have demonstrated that genes 5 and 6 of Gp C rotaviruses code for the structural protein VP 6 and the NS 34 equivalent of Gp A rotavirus, respectively [6, 27]. Thus, gene 5 and 6 cDNA clones were selected from a Cowden Gp C cDNA library and were developed for use as radiolabeled cDNA probes. The specificity of these two probes was examined against representative rotaviruses from different groups (A, B, or C), Gp C rotaviruses from different species, and distinct field strains of porcine Gp C rotaviruses in order to analyze the genetic diversity of genes 5 and 6 among Gp C rotaviruses and to develop cDNA probes for use as diagnostic and epidemiologic reagents.
Materials and methods Viruses
Human Gp C rotavirus strain 88-196 was purified from stool material collected from a 15-year-oldfemalewith diarrhea in Japan. BovineGp C rotavirus strain Shintoku, isolated from a diarrheic cow in Japan and adapted to growth in an MA 104 monkey kidney cell line [38], was purified from tissue culture material. Porcine Gp C rotavirus strain Cowden was purified from the intestinal contents of experimentally infected gnotobiotic pigs as previously described [-1]. Six porcine Gp C field strains were derived from the stools of diarrheic pigs in Ohio (NB, WH, Ah, HF, and KH) and Mississippi (Wi). An intestinal filtrate of each field strain was passaged in gnotobiotic pigs and the intestinal contents collectedasepticallyat the onset of diarrhea [36]. Two additional porcine rotavirus strains were included in the study: Gottfried (Gp A), propagated in MA 104 cells, and Ohio (Gp B), passaged in gnotobiotic pigs [37]. An uninfectedMA 104 cell extract and a fecal sample from an uninfected gnotobiotic pig were used as controls.
Genetic diversity of genes 5 and 6 among group C rotaviruses
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Preparation of viral dsRNA Samples containing virus particles were homogenized in Tris-CaC12 buffer (0.05 M Tris, 0.1 M NaC1, 1.0 mM CaC12, pH 7.5). The mixture was clarified by centrifugation at 8.700 x g and the resulting supernatant was centrifuged through a 30% (w/v) sucrose cushion at 107.170 x g for 2 h at 4 °C. The virus pellet was suspended in Tris-sodium chloride-EDTA buffer (0.01M Tris, 0.1M NaC1, 5raM EDTA, pH8.0) and SDS was added to a final concentration of 1%. The mixture was extracted with phenol-chloroform and the dsRNA precipitated with 2 volumes of ethanol overnight at - 20 °C. The concentration of nucleic acid was calculated from the absorbance at 260 nm. The two negative control samples were processed in the same manner.
Preparation of eDNA probes The generation and characterization of the cDNA clones of the Cowden genes 5 and 6 used in this study have been described [27]. Clone PC 4-19 consisted of nucleotides 356 to 1,352 of the Cowden gene 5 and clone CA2-19 consisted of nucleotides 115 to 1,145 of the Cowden gene 6 inserted into plasmid pTZ 18 R (Pharmacia LKB Biot~hnology Inc., Piscataway, N J). The recombinant DNA was nick-translated (Bethesda Research Laboratories. Inc., Gaithersburg, MD) using [32 p] dCTP (ICN Biochemicals Inc., Irvine, CA). The unincorporated nucleotides were removed by centrifugation through a Sephadex G-50 column [33].
RNA electrophoresis Viral dsRNA (1 to 5 gg per lane) was resolved in a 10% polyacrylamide slab gel (0.75 mm thick) using Laemmli's discontinuous buffer system [15]. Electrophoresis was conducted at 10 to 12mA for 14h. The RNA bands were visualized by ethidium bromide staining (0.5 gg/ml).
Northern and dot blot hybridization Northern blot hybridization was conducted using a method previously described [35]. Briefly, viral RNA in potyacrylamide gels was denatured in 0.1 M NaOH for 20 rain. The soaked gels were washed twice in 4 x TAE (1 x is 0.04 M Tris-acetate and 0.001 M EDTA) and once in 1 x TAE. The denatured RNA was then electro-transferred onto Nytran membranes (Schleicher & Schuell, Keene, NH) and immobilized by exposure to UV light. Hybridization conditions were similar to those previously described [29]. Prehybridization was for 4h at 42 °C in a solution containing 50% formamide, 5 x SSC, 50mM phosphate buffer (pH6.5), 0.2% SDS, 2 x Denhardt's solution, and 100gg/ml of yeast tRNA. Unless otherwise stated, all hybridizations were performed at 42 °C for 16 to 24 h in a similar solution with the addition of 4.5% dextran sulfate and the radiolabeled DNA probe (10 6 CPM/ml; specific activity, 650 Ci/mmote). Subsequently, the membranes were washed four times at room temperature in 2 x SSC, 0.1% SDS and then twice at 42 °C in 0.4 x SSC, 0.1% SDS. Finally, the membranes were exposed to Kodak diagnostic films (X-Omat RP-1; Eastman Kodak Company, Rochester, NY) at - 70°C for 1 to 6 days. For dot hypridization, heat-denatured RNA (0.5 to 1.0 gg) was dotted onto the Nytran membranes. The hybridization assay was conducted as described above.
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Jiang et al.: Genetic diversity of genes 5 and 6 among group C rotaviruses Results
Northern blot hybridization of Cowden strain gene 5 and 6 cDNA probes with Gp A, B and C rotaviruses The group specificity of the cDNA probes was examined by Northern blot hybridization. The distinct RNA electropherotypes of Gp A, B and C rotaviruses can be seen in Fig. 1 a and c. The Cowden Gp C gene 5 and 6 probes each recognized the corresponding gene from the Cowden strain, the excised homologous cDNA insert and the pTZ 18 R plasmid (Fig. 1 b and d, lanes 1 and 2). Neither probe hybridized with dsRNA from the Gp A or B rotaviruses under both moderate stringency conditions (50% formamide, 5 x SSC and 42 °C) (Fig. 1 b and d, lanes 3 and 4) and low stringency conditions (25% formamide, 5 x SSC, and 42 °C) (data not shown).
Northern blot hybridization analysis of Cowden gene 5 and 6 probes with Gp C rotaviruses from different species Northern blot hybridization was utilized to examine the species specificity of the gene 5 and 6 probes with Gp C rotaviruses from pigs, cows and humans. The similarities of RNA electropherotypes from Gp C rotaviruses of human and animal origin are seen in Fig. 2 a and c. In addition to hybridization with the excised homologous cDNA insert and the plasmid (Fig. 2b, lane 1), the gene 5 probe reacted with genomic segment 5 of Gp C rotaviruses from all three species, although relatively weaker signals were observed against the bovine and human Gp C rotaviruses (Fig. 2 b, lanes 2-4). In contrast, the gene 6 probe reacted strongly with the homologous Cowden gene 6, but signals were hardly detected with the gene 6 of bovine and human Gp C rotaviruses (Fig. 2 d, lanes 2-4) (weak signals could be seen after prolonged autoradiography; data not shown). However, at the low stringency conditions, hybridization signals were apparent with gene 6 of bovine and human Gp C rotaviruses (data not shown).
Northern blot hybridization of Cowden strain gene 5 and 6 probes against field strains of porcine Gp C rotavirus The strain specificity of the gene 5 and 6 probes was examined by Northern blot hybridization. In comparison with the prototyope Cowden strain, variation in the RNA electrophoretic migration patterns of the six field strains is seen although the overall RNA etectropherotype pattern is characteristic for Gp C rotavirus (Fig. 3 a and c). Each probe hybridized with the corresponding gene 5 or 6 of all field strains tested. However, the intensity of the signals on the autoradiogram varied. For example, the gene 5 probe showed strong signals with the gene 5 of the Cowden, NB, and Ah strains, moderate signals with the WH, HF, and Wi strains, and a weak signal with the KH strain (Fig. 3 b). With the gene 6 probe, strong signals were seen with the gene 6 of the Cowden, WH,
Fig. 1. Northern blot hybridization analysis of Cowden strain gene 5 (clone PC4-19) and 6 (clone CA 2-19) cDNA probes against representative porcine Gp A and B rotaviruses. Viral dsRNA was resolved in 10% polyacrylamide gel using Laemmli's discontinuous buffers and transferred onto Nytran membranes as described in the text. The hybridization was conducted at moderate stringency conditions (50% formamide, 5 x SSC and 42 °C). a and c Ethidium bromide-stained plasmid DNA and cDNA inserts (1 eDNA inserts were excised from recombinant DNA by digesting with EcoRI and BamHI and separated from the plasmid DNA), and RNA electropherotypes of Gp C (2), A (3) and B (4) rotaviruses. b and d The corresponding autoradiogram using the 32P-labeled gene 5 and 6 probes as indicated
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B.M. Jiang et al.
Fig. 2. Northern blot hybridization analysis of Cowden strain gene 5 and 6 probes against Gp C rotaviruses from different species. Samples were analyzed as described in the legend to Fig. 1. a and e Ethidium bromide-stained plasmid DNA and cDNA inserts (1), and RNA electropherotypes of porcine (2), bovine (3) and human (4) Gp C rotaviruses, b and d The corresponding autoradiogram using the 32P-labeled gene 5 and 6 probes as indicated
and Wi strains, moderate signals with the NB and K H strains, and weak signals with the Ah and H F strains (Fig. 3 d). Some weak signals could be attributed to lower d s R N A concentrations (e.g., the gene 5 probe against the K H strain), whereas other weak signals could not be attributed to lower R N A concentrations (e.g., the gene 5 probe against the W H strain).
Genetic diversity of genes 5 and 6 among group C rotaviruses
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Fig. 3. Northern blot hybridization of porcine Gp C Cowden strain gene 5 and 6 probes against field strains of porcine Gp C rotavirus. Samples were analyzed as in Fig. 1 a and e Ethidium bromide-stained plasmid DNA and cDNA inserts (DNA), and RNA migration patterns of the Cowden (Co) strain and 6 field strains (NB, WH, Ah, HF, KH, and Wi). b and d The corresponding autoradiogram using the 32P-labeled gene 5 and 6 probes as indicated
Analysis by dot hybridization The results of d o t hybridization assays were similar to N o r t h e r n blot hybridization using the same probes and stringency conditions. The gene 5 and 6 probes each hybridized with d s R N A from the h o m o l o g o u s G p C C o w d e n strain, but neither hybridized with d s R N A from the G p A Gottfried strain or the G p
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B Ohio strain (Fig. 4, 1 and 1 a). Against Gp C rotaviruses from three different species (porcine, bovine, and human), both probes recognized the homologous and heterologous viruses (Fig. 4, 2 and 2 a). However, similar to the Northern blot hybridization, much weaker signals were detected against the heterologous Shintoku and 88-196 strains with the gene 6 probe (Fig. 4, 2 a). Both probes hybridized with d s R N A from all six Gp C field strains in the dot blots. However, a variation in the intensities of the signals on the autoradiogram was observed. For example, weak signals were detected with the gene 5 probe against the H F and KH strains and with the gene 6 probe against the Ah and H F strains (Fig. 4, 3 and 3 a). Both probes hybridized strongly with the corresponding
Fig. 4. Dot hybridization analysis of Cowden gene 5 (1-3) and 6 (1 a-3 a) probes against Gp C, A, and B rotaviruses (1, i a); porcine, bovine, and human Gp C rotaviruses (2, 2 a); and homologous (Cowden) and heterologous strains of porcine Gp C rotaviruses designated NB, WH, Ah, HF, KH, and Wi (3, 3 a). The positive (recombinant DNA PC 4-19 or CA 219) and negative (MA 104 cells and porcine fecal specimen) controls are also included. Samples were dotted onto Nytran membranes and hybridized with 32P-labeled gene 5 and 6 probes as described in the text
Genetic diversity of genes 5 and 6 among group C rotaviruses
53
undigested recombinant DNA, but no signal was detected against an uninfected MA 104 cell extract or a negative porcine fecal specimen (Fig. 4, 1 and 1 a). The sensitivity of the two probes was investigated by dot hybridization against serial dilutions of Cowden dsRNA. Each probe was capable of detecting as low as 7.8 ng of RNA (data not shown). Discussion
Several criteria have been established to define rotavirus groups, including lack of serologic cross-reactivities among group antigens using heterologous antisera, RNA etectropherotyping and terminal fingerprinting of individual genomic segments [22, 23, 30]. Hybridization assays have been successfully used in the diagnosis of Gp A rotavirus infections, the analysis of the genetic relatedness among Gp A rotaviruses and the differentiation of Gp A from non Gp A rotaviruses [8, 9, 12, 16, 20, 29]. Further, cDNA probes derived from the gene encoding VP 6 (gene 6) of the Gp A Wa (subgroup II) and SA 11 (subgroup I) strains were used for differentiating Gp A rotaviruses into subgroups [ 17]. This genetic approach for subgrouping Gp A rotaviruses correlated with serologic analysis using subgroup-specific monoclonal antibodies. The cDNA gene 5 probe developed here proved to be Gp C rotavirus-specific, while reacting broadly among Gp C rotaviruses from different species. The variable reactivities of our gene 5 probe against dsRNA of similar quantity among field strains of porcine Gp C rotaviruses might reflect subgroup differences. However, no antigenic evidence for subgroup differences among Gp C rotaviruses has yet been reported. The gene 6 probe successfully recognized several distinct porcine Gp C rotavirus field strains, while demonstrating Gp C rotavirus-specificity. An interesting observation in this study was the apparently greater genetic diversity among the gene 6 of Gp C rotavirus strains from different species as detected by the intensity of the hybridization signal on the autoradiogram. Similar studies of genetic diversity among Gp A rotaviruses from different species using cDNA probes to genes encoding a nonstructural protein have not been reported, although evidence for genetic diversity in a gene encoding a nonstructural protein (gene 5) was found among human Gp A rotaviruses in RNA-RNA hybridization studies [34]. The biological significance of the genetic variation among genes 5 and 6 of Gp C strains requires further analysis of additional strains. Recently, the genetic relatedness of human (88-220 strain) and porcine (Cowden strain) Gp C rotaviruses was examined by Northern blot hybridization analysis using ssRNA probes, and strong homology was observed in most of the 11 gene segments [25]. Our findings are consistent with that report. Our previous study reported the genetic relatedness of the gene encoding the outer capsid protein (VP 7) between porcine (Cowden strain) and bovine (Shintoku strain) Gp C rotaviruses using the VP 7 cDNA probe [14]. This study further demonstrated the genetic relatedness of genes 5 and 6 between porcine and
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bovine Gp C rotaviruses. The identification of a group C rotavirus in cattle indicates that, analogous to Gp A rotaviruses, Gp C rotaviruses m a y exhibit a broad host range. The unavailability of reagents for the identification of Gp C rotaviruses a n d the inability to serially p r o p a g a t e m o s t strains in cell culture have h a m p e r e d epidemiologicat surveys. In this study, we report the development o f hybridization assays for the identification o f G p C rotaviruses using the C o w d e n gene 5 and 6 c D N A probes. These assays should provide sensitive and specific m e t h o d s for the diagnosis of G p C rotavirus infections in h u m a n s a n d animals and should facilitate epidemiological studies. However, for large scale epidemiologic studies, nonradiolabeled probes m a y be preferable for routine use. Acknowledgements
We thank A. V. Parwani and B. I. Rosen for assistance in the hybridization assays. This study was supported in part by special research grant #89-34116-4625 from the U.S. Department of Agriculture, Cooperative State Research Service. Salaries and research support were provided by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Journal article No. 66-91. References
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Authors' address: Linda J. Saif, Ohio Agricultural Research and Development Center, The Ohio State University, 1680 Madison Avenue, Wooster, OH 44691-4096, U.S.A. Received November 24, 1991
